Oxytocin treatment to modify blood glucose and treat metabolic disease

ABSTRACT

The disclosure provides a method for enhancing memory of a subject, lowering blood glucose levels in a subject, and treating schizophrenia, the methods comprise intranasally administering to the subject an amount of a oxytocin peptide or analog thereof having a beneficial effect.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/812,850, filed May 7, 2013, which is a U.S. National StageApplication filed under 35 U.S.C. § 371 and claims priority toInternational Application No. PCT/US2011/046046, filed Jul. 30, 2011,which application claims priority under 35 U.S.C. § 119 to U.S.Provisional Application Ser. No. 61/369,607, filed Jul. 30, 2010. Thedisclosures of which are incorporated herein by reference.

BACKGROUND

Oxytocin and its receptors exist in areas of the brain implicated in thesymptoms of schizophrenia such as the nucleus accumbens and thehippocampus. Previous studies have shown that oxytocin administered tohumans has a propensity to decrease verbal memory. Studies that haveadministered a single dose of oxytocin worsened recall for words.(Ferrier et al., Life Sci., 27(24), 1980; Fehm-Wolfsdorf et al.,Pscyhoneuroendrocrinology, 9(3):285-92, 1984; Bruins et al., Peptides,13(3):461-8, 1991; Heinrichs et al., Phsiol. Behav, 83(1):31-8, 2004).In fact one study gave a person with obsessive compulsive disorderintranasal oxytocin for 4 weeks and found that it produced significantworsening of his memory (Ansseau et al, 1987).

SUMMARY

The disclosure provides a method for enhancing memory of a subject, themethod comprising intranasally administering to the subject an amount ofan oxytocin peptide or analogue thereof wherein the amount of oxytocinpeptide or analogue thereof improves a subject's memory compared to thesubject before administration of the oxytocin peptide or analoguethereof.

The disclosure also provides a method for reducing blood glucose levelsof a subject, the method comprising intranasally administering to thesubject an amount of a oxytocin peptide or analogue thereof wherein theamount of oxytocin peptide or analogue thereof reduces a subject's bloodglucose compared to the subject's blood glucose before administration ofthe oxytocin peptide or analogue thereof.

The disclosure provides a method of treating schizophrenia comprisingadministering intranasally, as an adjunctive therapy, oxytocin or ananalogue thereof.

The disclosure provide methods of using an oxytocin formulation forimproving psychiatric disorders, memory and blood glucose levels. Thedisclosure provides use of a formulation for intranasal delivery of anoxytocin peptide chronically for the treatment of a disease or disorderselected from the group consisting of a psychiatric disease or disorder,a metabolic disease or disorder and a memory disease or disorder. In oneembodiment, the formulation is delivered at least once per day at a doseof about 20-100 IU. In another embodiment, the dose is at least twotimes per day at a dose of about 20-100 IU per administration. In yetanother embodiment, the dose is about 40-80 IU per day. In oneembodiment, the psychiatric disease or disorder is schizophrenia. Inanother embodiment, the metabolic disease or disorder is diabetes. Inyet another embodiment, the use improves non-social memory.

The disclosure also provides a method for enhancing non-social memory ina subject, the method comprising intranasally administering to thesubject an amount of a oxytocin peptide or analogue thereof in an amountof about 20-100 IU per day, wherein the amount of oxytocin peptide oranalogue thereof improves a subject's non-social memory compared to thesubject before administration of the oxytocin peptide or analoguethereof. In one embodiment, the subject has a memory disease ordisorder. In yet another embodiment, the subject has a psychiatricdisease or disorder that can be improved by improving memory. In yetanother embodiment, the subject has schizophrenia. In anotherembodiment, the oxytocin peptide or analogue is administered at leasttwice per day. In yet another embodiment, the oxytocin peptide oranalogue is administered two-times per day. In a further embodiment, thetotal daily dose is about 80-100 IU per day.

The disclosure also provides a method of treating a metabolic disorderin a subject, comprising intranasally administering an oxytocin peptideintranasally at a dose of about 20-100 IU per day. In one embodiment,the metabolic disorder is selected from the group consisting of diabetesobesity, hyperlipemia, diabetes, fatty liver, hypertension, andcardiovascular disease.

The disclosure also provides a method for reducing blood glucose levelsof a subject, the method comprising intranasally administering to thesubject an amount of a oxytocin peptide or analogue thereof wherein theamount of oxytocin peptide or analogue thereof reduces a subject's bloodglucose compared to the subject's blood glucose before administration ofthe oxytocin peptide or analogue thereof.

In any of the foregoing embodiment, the oxytocin peptide or analoguethereof is a peptide with at least about 75%, 80%, 85%, 90%, 95%, 97% or99% identity to SEQ ID NO:1 and wherein the oxytocin peptide or analoguethereof improves memory or reduces blood glucose.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide” includesa plurality of such peptides and reference to “the cell” includesreference to one or more cells known to those skilled in the art, and soforth.

Also, the use of “and” means “and/or” unless stated otherwise.Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Oxytocin is a nine amino acid cyclic peptide hormone with two cysteineresidues that form a disulfide bridge between positions 1 and 6. Humanoxytocin comprises the sequence Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQID NO:1). Oxytocin is released from the pituitary gland and stimulatesthe contraction of smooth muscle of the uterus during labor andfacilitates release of milk from the breast during nursing. Oxytocin hashistorically been used to induce labor.

As used herein, “oxytocin” or “oxytocin peptide” refers to a substancehaving biological activity associated with natural oxytocin. Oxytocin oroxytocin peptide can be a naturally occurring endogenous peptide,fragments, analogues or derivatives thereof. Oxytocin or oxytocinpeptide can also be a non-endogenous peptide, fragments, analogues orderivatives thereof. An oxytocin peptide includes both natural orsynthetic, therapeutically or prophylactically active, peptidefragments, peptide analogues, and chemically modified derivatives orsalts of active peptides. There are processes described for theproduction of oxytocin, see for example U.S. Pat. Nos. 2,938,891 and3,076,797. In addition, oxytocin is commercially available. A variety ofpeptide analogues and derivatives are available and others can becontemplated for use within the disclosure and can be produced andtested for biological activity according to known methods. Oxytocinanalogues may included, but are not limited to,4-threonine-1-hydroxy-deaminooxytocin, 4-serine, 8-isoleucine-oxytocin,9-deamidooxytocin, 7-D-proline-oxytocin and its deamino analog,(2,4-diisoleucine)-oxytocin, deamino oxytocin analog,1-deamino-1-monocarba-E12-Tyr(OMe)]-OT(dCOMOT), carbetocin, 4-threonine,7-glycine-oxytocin (TG-OT), oxypressin, deamino-6-carba-oxytoxin (dC60),deamino-1 monocarba-(2-O-methyltyrosine)-oxytocin [d(COMOT)]);[Thr4-Gly7]-oxytocin (TG-OT); oxypressin; Ile-conopressin; atosiban;deamino-6-carba-oxytoxin (dC60), d[Lys(8)(5/6C-Fluorescein)]VT,d[Thr(4), Lys(8)(5/6C-Fluorescein)]VT,[HO(1)][Lys(8)(5/6C-Fluorescein)]VT, [HO(1)][Thr(4),Lys(8)(5/6CFluorescein)]VT, d[Om(8)(5/6C-Fluorescein)]VT, d[Thr(4),Om(8)(5/6C-Fluorescein)]VT, [HO(1)][Om(8)(5/6C-Fluorescein)]VT,[HO(I)][Thr(4), Om(8)(5/6C-Fluorescein)]VT, desmopressin, and1-deamino-oxytocin in which the disulfide bridge between residues 1 and6 is replaced by a thioether, L-371,257 and the related series ofcompounds containing an ortho-trigluoroethoxyphenylacetyl core such asL-374,943. Oxytocin peptide and polypeptide useful in the methods andcompositions of the disclosure include peptides that are obtainable bypartial substitution, addition, or deletion of amino acids within anaturally occurring or native peptide sequence.

As used herein, “analogues and derivatives” refers to any peptideanalogous of naturally occurring oxytocin wherein one or more aminoacids within the peptide have been substituted, deleted, or inserted.The term also refers to any peptide wherein one or more amino acids havebeen modified, for example by chemical modification. In general, theterm covers all peptides which exhibit oxytocin activity but which may,if desired, have a different potency or pharmacological profile.Peptides can be chemically modified, for example, by amidation of thecarboxyl terminus (—NH₂), the use of D amino acids in the peptide,incorporation of small non-peptidyl moieties, as well as themodification of the amino acids themselves (e.g., alkylation oresterification of side chain R-groups). Such analogues, derivatives andfragments should substantially retain the desired biological activity ofthe native oxytocin peptide.

In still other embodiments the oxytocin analogs are fragments ofoxytocin, for example, peptide cleavage products. Such fragments may bechemically synthesized or derived by any known means. Oxytocin fragmentsof the disclosure retain bioactivity similar to or greater thanoxytocin. Such fragments may be capable of crossing the blood brainbarrier.

In another embodiment of the disclosure, oxytocin analogs are syntheticoxytocin molecules that retain oxytocin bioactivity. Such analogmolecules are capable of acting in a manner similar to endogenousoxytocin, including binding the oxytocin receptor. Analogs of this typemay be derivatives of oxytocin or have completely new molecularstructures.

In another embodiment oxytocin analogs can be modified for increasedstability, enhancement of transport across the blood brain barrier,retention in the brain once they have crossed the blood brain barrier ora combination of the foregoing. Modifications to increase stability andenhance blood brain barrier transport may include, but are not limitedto, esterification with steroids, such as cholesteryl, or esterificationwith fatty alcohols, such as C-8 to C-22 alcohols. Modifications toincrease retention in the brain include, but are not limited to,covalent attachment of 1,4-dihydrotrigonellinate and other redoxsensitive functionalities, such as quinones and derivatives such asbenzoquinones, naphthoquinones, indolequinones, nitroheterocycles suchas nitrobenzyl, nitrofurans, and nitroimadzole derivatives.

The peptides/polypeptides described and/or contemplated herein can beprepared by chemical synthesis using either automated or manual solidphase synthetic technologies, generally known in the art. The peptidescan also be prepared using molecular recombinant techniques known in theart.

A polypeptide or peptide of the disclosure may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant polypeptide. The resulting expressed polypeptide may then bepurified from such culture (e.g., from culture medium or cell extracts)using known purification processes, such as gel filtration and ionexchange chromatography. The purification of a polypeptide may alsoinclude an affinity column containing agents which will bind to thepolypeptide; one or more column steps over such affinity resins asconcanavalin A-agarose, Heparin-Toyopearl or Cibacrom blue 3GASepharose; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography. Alternatively, a polypeptide ofthe disclosure may also be expressed in a form that will facilitatepurification. For example, it may be expressed as a fusion polypeptide,such as those of maltose binding polypeptide (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion polypeptides are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and InVitrogen, respectively. A polypeptide can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (“Flag”) iscommercially available. Finally, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media, e.g., silica gel having pendant methyl or other aliphaticgroups, can be employed to further purify the polypeptide. Some or allof the foregoing purification steps, in various combinations, can alsobe employed to provide a substantially homogeneous isolated recombinantpolypeptide. A polypeptide thus purified is substantially free of othermammalian polypeptides and is defined in accordance with the disclosureas a “substantially purified polypeptide. A polypeptide of thedisclosure may also be expressed as a product of transgenic animals,e.g., as a component of the milk of transgenic cows, goats, pigs, orsheep which are characterized by somatic or germ cells containing apolynucleotide encoding the polypeptide.

It is also possible to utilize an affinity column comprising amonoclonal antibody generated against an oxytocin peptide of thedisclosure, to affinity-purify an expressed polypeptide. Thesepolypeptides can be removed from an affinity column using conventionaltechniques, e.g., in a high salt elution buffer and then dialyzed into alower salt buffer for use or by changing pH or other componentsdepending on the affinity matrix utilized, or be competitively removedusing the naturally occurring substrate of the affinity moiety, such asa polypeptide derived from the disclosure. In this embodiment of thedisclosure, an anti-polypeptide antibody of the disclosure or otherpolypeptides that can interact with a polypeptide of the disclosure, canbe bound to a solid phase support such as a column chromatography matrixor a similar substrate suitable for identifying, separating, orpurifying cells that express polypeptides of the disclosure on theirsurface.

A polypeptide may also be produced by known conventional chemicalsynthesis. Methods for constructing polypeptides of the disclosure bysynthetic means are known to those skilled in the art. The syntheticallyconstructed polypeptides, by virtue of sharing primary, secondary ortertiary structural and/or conformational characteristics with nativepolypeptides may possess biological properties in common therewith,including polypeptide activity. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedpolypeptides in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The desired degree of purity depends on the intended use of apolypeptide. A relatively high degree of purity is desired when apolypeptide is to be administered in vivo, for example. In such a case,polypeptides are purified such that no polypeptide bands correspondingto other polypeptides are detectable upon analysis by SDS-polyacrylamidegel electrophoresis (SDS-PAGE). It will be recognized by one skilled inthe pertinent field that multiple bands corresponding to the polypeptidecan be visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. A polypeptideof the disclosure is purified to substantial homogeneity, as indicatedby a single polypeptide band upon analysis by SDS-PAGE. The polypeptideband can be visualized by silver staining, Coomassie blue staining, or(if the polypeptide is radiolabeled) by autoradiography.

Schizophrenia is a crippling, clinically heterogeneous disease thatafflicts patients with hallucinations and delusions as well as profoundimpairments in social and executive function. Current medicationtreatments for schizophrenia, though they significantly alleviate someof these symptoms, fall short of the goal of remission in a largemajority of cases. As such, finding novel treatments that impact thebroad range of symptoms of schizophrenia is an urgent priority.

In particular, deficits in cognitive function—a particularly disablingcomponent of schizophrenia-are at best partially ameliorated withcurrent antipsychotic medications. This shortcoming is notable, as thesedeficits often predict patient's level of dysfunction. Cognitivedeficits include problems with attention and goal-initiation, as well asimpairments in several types of memory. Though there have been effortsto develop psychopharmacologic treatments to address this cluster ofsymptoms, no current antipsychotic medications decisively improve thisdomain of function.

The disclosure provides a method of treating schizophrenia comprisingdelivering oxytocin peptide intranasally to a subject. The oxytocinpeptide may be a naturally occurring purified form of oxytocin, arecombinant for of oxytocin and analog of oxytocin, a chemicallymodified form of oxytocin or a combination thereof. The intranasaldelivery improves uptake, compliance and treatment of schizophrenia. Inone embodiment, the method comprises delivery an oxytocin peptideintranasally at least twice per day. In another embodiment, the oxytocinpeptide is delivered at least twice per day at a dose of about 20-50 IU(international units) per administration (e.g., about 40-100 IU perday). Doses may range from about 10-80 IU per administration and willdepend upon various factors readily identifiable to a physician (e.g.,body weight, route of administration, formulation, severity of a diseaseor disorder and the like). Accordingly, the total dose for a subject maybe about 20-160 (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150 or 160) IU per day. In another embodiment, an oxytocinpeptide is administered intranasally at a dose of about 40-100 IU perday. The dosing may be one or more times per day. The dosing maycontinue for several days, weeks, months or years. In anotherembodiment, the oxytocin peptide is administered as an adjunctivetherapy to standard/current therapy for schizophrenia. In yet anotherembodiment, the oxytocin peptide is administered chronically or longterm for at least 3 weeks or longer at least twice a day as describedabove. In yet other embodiments, the oxytocin peptide may beadministered intraperitoneally, intravascularly, intramuscularly,orally, and the like, in either delayed or sustained releaseformulations or immediate release formulations.

The disclosure also provides a method of treating memory loss, memoryfunction, recall, memorization, improvement in memory in Alzheimer'sdisease or other memory function disorder by administering to a subjectin need of such treatment an effective amount of an oxytocin peptide. Inone embodiment, the subject is a subject presenting with characteristicsschizophrenia or having schizophrenia. The oxytocin peptide may be anaturally occurring purified form of oxytocin, a recombinant for ofoxytocin and analog of oxytocin, a chemically modified form of oxytocinor a combination thereof. In one embodiment, the oxytocin peptide isadministered intranasally, mucosally, sublingually and the like. In oneembodiment, the method comprises delivery an oxytocin peptideintranasally at least twice per day. In another embodiment, the oxytocinpeptide is delivered at least twice per day at a dose of about 20-40 IU(international units) per administration (e.g., about 40-100 IU perday). In another embodiment, an oxytocin peptide is administeredintranasally at a dose of about 40-100 IU per day. The dosing may be oneor more times per day. The dosing may continue for several days, weeks,months or years. In yet another embodiment, the oxytocin peptide isadministered chronically or long term for at least 3 weeks or longer atleast twice a day as described above. In yet other embodiments, theoxytocin peptide may be administered intraperitoneally, intravascularly,intramuscularly, orally, and the like, in either delayed or sustainedrelease formulations or immediate release formulations.

Impairments in cognitive performance and memory have been reported inhumans treated with oxytocin, accordingly, the data presented herein isunexpected. To examine memory function two memory tasks, the CaliforniaVerbal Learning Test (CVLT) and Letter Number Sequencing (LNS) task fromthe WAIS-III, were used to examine the potential effect on memory andlearning. The disclosure demonstrates that oxytocin peptideadministration intranasally at least twice per day (e.g., 20-40 IU peradministration) as a primary therapy or adjunctive therapy improvesnon-social memory (e.g., non-socially relevant memory) in subjects. Thisis unexpected. Prior research studies have found that oxytocin had noeffect or worsening of word memory devoid of social relevance. It hasbeen recognized that oxytocin is a peptide that regulates socialaffiliation thus it is the role of oxytocin for enhancing socialrecognition and bonding is expected. However, the role of oxytocin innon-social relevant memory is totally unexpected based upon the priorresearch.

As used herein socially relevant memory would be memory forsocially-relevant stimuli. Socially relevant stimuli would be anystimuli that have a socially relevant connotation. For example, sociallyrelevant stimuli include human faces, words such as “love” or “hate”.Socially relevant stimuli include stimuli that include an emotionalstate of bonding between humans. In contrast socially neutral stimuliwould include a non-social object such as table, or words such asenvelope, sky, numbers and the like. The disclosure provides methods ofimproving word memory and non-human object memory.

Thus, the methods of the disclosure comprise treating a mammal includinga human with an amount of oxytocin peptide that stimulates memoryfunction or treats a disease associated with memory inhibition orreduction. Further, the disclosure provides methods and composition fortreating psychiatric disorders including schizophrenia. In oneembodiment, the methods comprise administering an oxytocin peptideintranasally. In another embodiment, the method comprises administeringthe oxytocin mucosally such as sublingually. In yet other embodiments,the oxytocin peptide may be administered intraperitoneally,intravascularly, intramuscularly, orally, and the like, in eitherdelayed or sustained release formulations or immediate releaseformulations.

The effect or an effective dose of oxytocin peptide can be measuredusing various recognized testing methods. For example, the CVLT-II is acommonly-used test of new learning and declarative verbal memory, whichclosely resembles the HVLT (Hopkins Verbal Learning Test), a part of thestandardized MATRICS cognitive battery, now used to measure cognitivechanges in Schizophrenia clinical trials. In the CVLT-II, a list of 16words (List A) from various semantic categories are read over 5 trials.After each trial, subject is asked to recall as many words as they can(free recall). An interference list (List B) is then presented. Free andcued recall of List A is measured immediately following (short delay)and 20 minutes (long delay). At the end of the test, a 44 wordrecognition task is performed with subjects identifying target wordsamong 28 distractors. The CVLT's format allows assessment of multipleaspects of cognition: overall recall ability, rate of learning oversequential trials, ability to retain learned material, and itemrecognition.

The Letter Number Sequencing subtest (LNS) is a component of the largerWAIS-III and is used to measure attention and working memory. It issimilar to the Letter number span test, which is also part of theMATRICS. In each trial of the LNS, a list of letters and numbers areread in a mixed order to participants; participants are asked to recallfirst the digits and then the letters in the order they were presented.Length of the list ranges from 2 to 8; each length is tested threetimes. Scores are calculated by totaling the correct responses for eachlength.

A psychiatric or neurological disorder that may be treated by themethods of the disclosure include pervasive developmental disorder nototherwise specified, non-verbal learning disabilities, autism and autismspectrum disorders, attention deficit hyperactivity disorder (ADHD),anxiety disorders, Post-traumatic stress disorders, social phobia,generalized anxiety disorder, social deficit disorders, schizotypalpersonality disorder, schizoid personality disorder, schizophrenia,cognitive deficit disorders, dementia, Alzheimer's and other memorydeficit disorders. In the methods of the disclosure such psychiatric orneurological disorders may be treated by administering an oxytocinpeptide intranasally at least twice per day at a dose of about 20-40 IUper administration. In another embodiment, the dosing is 40 IU peradministration or about 100 IU per day. In another embodiment, thedosing is continued for several days, weeks or years. In yet anotherembodiment, the psychiatric or neurological disorder is treated byimproving non-social relevant memory capabilities. In yet otherembodiments, the oxytocin peptide may be administered intraperitoneally,intravascularly, intramuscularly, orally, and the like, in eitherdelayed or sustained release formulations or immediate releaseformulations.

The disclosure also provides a method of improving non-social memorycapabilities in a normal subject, wherein the memory of the subjecttreated with an oxytocin peptide is improved compared to the samesubject or a different subject not treated with oxytocin. In thisembodiment of the disclosure a subject is treated to improve non-socialmemory by administering an oxytocin peptide intranasally at a dose ofabout 40-100 IU per day. The dosing may be one or more times per day.The dosing may continue for several days, weeks, months or years. In yetother embodiments, the oxytocin peptide may be administeredintraperitoneally, intravascularly, intramuscularly, orally, and thelike, in either delayed or sustained release formulations or immediaterelease formulations.

The disclosure also provides a method of treating metabolic disorderscomprising intranasally delivering oxytocin peptide to a subject. Theoxytocin peptide may be a naturally occurring purified form of oxytocin,a recombinant for of oxytocin and analog of oxytocin, a chemicallymodified form of oxytocin or a combination thereof. In one embodiment,the oxytocin peptide is administered intranasally, mucosally,sublingually and the like. The administration results in a reduction ofblood glucose and weight loss. Metabolic diseases and disorders that maybe treated by the methods of the disclosure include diabetes andobesity. By “treat” means reduction or modulation of a symptom of thedisease. For example, administration of oxytocin intranasally can resultin a reduction of blood glucose in diabetic patients or a reduction ofweight in obese subjects. Such administration is recurring and typicallylong term. In one embodiment, an oxytocin peptide is administeredintranasally at a dose of about 40-100 IU per day. The dosing may be oneor more times per day (e.g., 2, 3, 4, 5, 6 times per day). The dosingmay continue for several days, weeks, months or years. In yet otherembodiments, the oxytocin peptide may be administered intraperitoneally,intravascularly, intramuscularly, orally, and the like, in eitherdelayed or sustained release formulations or immediate releaseformulations.

Oxytocin peptides or their salts can be formulated for deliver byadmixture with pharmaceutically acceptable non-toxic excipients orcarriers. Mention may be made, as examples of pharmaceuticallyacceptable salts, of the addition salts with inorganic or organic acids(such as acetate, trifluoroacetate, propionate, succinate, benzoate,fumarate, maleate, oxalate, methanesulphonate, isethionate,theophyllinacetate, salicylate, methylenebis-.beta.-oxynaphthoate,hydrochloride, sulphate, nitrate and phosphate), the salts with alkalimetals (sodium, potassium or lithium) or with alkaline-earth metals(calcium or magnesium), the ammonium salt or the salts of nitrogenousbases (ethanolamine, trimethylamine, methylamine, piperidine,benzylamine, N-benzyl-.alpha.-phenethylamine, choline, arginine,leucine, lysine or N-methylglucamine).

The disclosure provides pharmaceutical compositions of oxytocin peptidesor their salts. The oxytocin peptide or their physiologically acceptablesalts or solvates, may be formulated for administration for injection,or for oral, topical, nasal, inhalation, insufflation (either throughthe mouth or the nose) buccal, parenteral, rectal administration orother forms of administration. The disclosure provides pharmaceuticalcompositions comprising effective amounts of an oxytocin peptidetogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants, excipients and/or carriers. Suchcompositions include diluents of various buffer content (e.g., Tris-HCl,acetate, phosphate), pH and ionic strength; additives such as detergentsand solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimerosal, benzyl alcohol) and bulking substances (e.g., lactose,mannitol).

The compositions may also be incorporated into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, andthe like or liposomes. Hyaluronic acid may also be used. Biocompatibleabsorbable polymers may be selected from the group consisting ofaliphatic polyesters, copolymers and blends, which include, but are notlimited to, homopolymers and copolymers of lactide (which include D-,L-, lactic acid and D-, L- and meso lactide), glycolide (includingglycolic acid), epsilon-caprolactone, p-dioxanone (1,4-dioxan-2-one),alkyl substituted derivatives of p-dioxanone (i.e.,6,6-dimethyl-1,4-dioxan-2-one), triethylene carbonate(1,3-dioxan-2-one), alkyl substituted derivatives of 1,3-dioxanone,delta-valerolactone, beta-butyrolactone, gamma-butyrolactone,epsilon-decala tone, hydroxybutyrate, hydroxyvalerate,1,4-dioxepan-2-one and its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14dione, 1,5-dioxepan-2-one, and polymer blends thereof.

Such compositions may influence physical state, stability, rate of invivo release, and rate of in vivo clearance. See, e.g., Remington sPharmaceutical Sciences, 18th ed., (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712). The compositions may be prepared in liquidform, or be in dried powder, such as lyophilized form.

Contemplated for use herein are oral solid dosage forms, which aredisclosed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosageforms include tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation may be used toformulate compositions. Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers. A description ofpossible solid dosage forms for the therapeutic is given by Marshall, K.In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter10, 1979). In general, the formulation will include an Oxytocin peptideand inert ingredients (which allow for protection against the stomachenvironment and release of the biologically active material in theintestine).

To ensure full gastric resistance a coating impermeable to at least pH5.0 is useful. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L3OD, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings that make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic, i.e., powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets may be thick starch or other ediblepaper. For pills, lozenges, molded tablets or tablet triturates, moistmassing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets. The formulationof the material for capsule administration can also be as a powder,lightly compressed plugs or even as tablets. The therapeutic can also beprepared by compression.

Colorants and flavoring agents may all be included. For example, thepeptide (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial or filler. These diluents or fillers can include carbohydrates,especially mannitol, anhydrous lactose, cellulose (e.g.,microcrystalline cellulose), sucrose, calcium hydrogen phosphatemodified dextrans and starch. Certain inorganic salts may be also beused as fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include, but arenot limited to, starch (e.g., potato starch or the commercialdisintegrant based on starch, Explotab). Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch (e.g., pregelatinised maize starch) and gelatin.Others include methyl cellulose (MC), ethyl cellulose (EC) andcarboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) andhydroxypropylmethyl cellulose (HPMC) can both be used in alcoholicsolutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes, talc and silica.Soluble lubricants may also be used such as sodium lauryl sulfate,magnesium lauryl sulfate, polyethylene glycol of various molecularweights, Carbowax 4000 and 6000.

Glidants that can improve the flow properties of the drug duringformulation and to aid rearrangement during compression can be added.The glidants can include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant can be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents can beused and can include benzalkonium chloride or benzethomium chloride. Thelist of potential non-ionic detergents that can be included in theformulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate,polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerolmonostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester,methyl cellulose and carboxymethyl cellulose. These surfactants can bepresent in the formulation of the protein or derivative either alone oras a mixture in different ratios.

Additives that potentially enhance uptake of the agent are, for example,the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release oral formulation may be desirable. The agent can beincorporated into an inert matrix that permits release by eitherdiffusion or leaching mechanisms, e.g., gums. Slowly degeneratingmatrices may also be incorporated into the formulation. Some entericcoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars that can be applied in a coating pan. The therapeutic agentcan also be given in a film coated tablet and the materials used in thisinstance are divided into two groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials can be used to provide the optimum film coating. Filmcoating may be carried out in a pan-coater or in a fluidized bed or bycompression coating.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

The Oxytocin peptides may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations disclosed previously, the oxytocinpeptides may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the Oxytocin peptides may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Toxicity and therapeutic efficacy of such oxytocin peptides can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animal/animal models (such as those described herein),e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.While compounds that exhibit toxic side effects may be used, care shouldbe taken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes theIC.sub.50 (i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

An oxytocin peptide and components of a therapeutic composition may beintroduced parenterally, topically, or transmucosally, e.g., orally,nasally, or rectally, or transdermally. Parenteral administrationincludes, for example, intravenous injection, intra-arteriole,intramuscular, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial administration.

Because many oxytocin peptide are capable of crossing the blood brainbarrier oxytocin peptides permit oral, parenteral or intravenousadministration. Alternatively, the agent can be modified or otherwisealtered so that it can cross or be transported across the blood brainbarrier. Many strategies known in the art are available for moleculescrossing the blood-brain barrier, including but not limited to,increasing the hydrophobic nature of a molecule; introducing themolecule as a conjugate to a carrier, such as transferring, targeted toa receptor in the blood-brain barrier, or to docosahexaenoic acid andthe like.

In another embodiment, an Oxytocin peptide may be administered bysurgical intervention including a procedure of drilling a small hole inthe skull to administer the agent.

In another embodiment, the molecule can be administered intracraniallyor intraventricularly. In another embodiment, osmotic disruption of theblood-brain barrier can be used to effect delivery of agent to the brain(Nilayer et al., Proc. Natl. Acad. Sci. USA 92:9829-9833 (1995)). In yetanother embodiment, an agent can be administered in a liposome targetedto the blood-brain barrier. Administration of pharmaceutical agents inliposomes is known (see Langer, Science 249:1527-1533 (1990); Treat etal., in Liposomes in the Therapy of infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. pp. 317-327 and353-365 (1989).

Some predictions have been made concerning the ability of molecules topass through the blood-brain barrier, the rate and extent of entry of anoxytocin peptide or a formulation comprising an oxytocin peptide intothe brain are generally considered to be determined by partitioncoefficient, ionization constant(s), and molecular size.

In another embodiment, a therapeutic formulation comprising an oxytocinpeptide can be delivered in a vesicle, in particular a liposome (seeLanger, Science 249:1527-1533 (1990); Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss: New York, pp. 317-327 and 353-365 (1989)).

In another embodiment, a therapeutic formulation comprising an oxytocinpeptide can be delivered in a controlled release system. For example,the oxytocin peptide may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPress: Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley: New York(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In addition, any of the materials described herein can be administeredto any part of the mammal's body including, without limitation, brain,spinal fluid, blood stream, lungs, nasal cavity, intestines, stomach,muscle tissues, skin, peritoneal cavity, and the like. Thus, an oxytocinpeptide (e.g., a neurotensin analog) can be administered by intravenous,intraperitoneal, intramuscular, subcutaneous, extracranial, intrathecal,and intradermal injection, by oral administration, by inhalation, or bygradual perfusion over time. For example, an aerosol preparation can begiven to a mammal by inhalation. It is noted that the duration oftreatment with the materials described herein can be any length of timefrom as short as one day to as long as a lifetime (e.g., many years).For example, a formulation comprising an oxytocin peptide can beadministered once (or twice, three times, etc.) daily, weekly, monthly,or yearly.

Preparations for administration can include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and injectable organic esters.Aqueous carriers include, without limitation, water as well as alcohol,saline, and buffered solutions. Preservatives, flavorings, and otheradditives such as, for example, antimicrobials, anti-oxidants, chelatingagents, inert gases, and the like may also be present.

An oxytocin peptide or a pharmaceutical composition comprising anoxytocin peptide may be dispensed intranasally or mucosally as apowdered or liquid nasal spray, suspension, nose drops, a gel, film orointment, through a tube or catheter, by syringe, by packtail, bypledget (a small flat absorbent pad), by nasal tampon or by submucosalinfusion. Nasal drug delivery can be carried out using devicesincluding, but not limited to, unit dose containers, pump sprays,droppers, squeeze bottles, airless and preservative-free sprays,nebulizers (devices used to change liquid medication to an aerosolparticulate form), metered dose inhalers, and pressurized metered doseinhalers. It is important that the delivery device protect the drug fromcontamination and chemical degradation. The device should also avoidleaching or absorption as well as provide an appropriate environment forstorage. Each drug needs to be evaluated to determine which nasal drugdelivery system is most appropriate. Nasal drug delivery systems areknown in the art and several are commercially available.

An oxytocin peptide or a pharmaceutical composition comprising anoxytocin peptide may be conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellantincluding, but not limited to, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons,compressed air, nitrogen or carbon dioxide. An aerosol system requiresthe propellant to be inert towards the pharmaceutical composition. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver an accurately metered amount.

The means to deliver an oxytocin peptide or pharmaceutical compositioncomprising an oxytocin peptide to the nasal cavity as a powder can be ina form such as microspheres delivered by a nasal insufflator device (adevice to blow a gas, powder, or vapor into a cavity of the body) orpressurized aerosol canister. The insufflator produces a finely dividedcloud of the dry powder or microspheres. The insufflator may be providedwith means to ensure administration of a substantially metered amount ofthe pharmaceutical composition. The powder or microspheres should beadministered in a dry, air-dispensable form. The powder or microspheresmay be used directly with an insufflator which is provided with a bottleor container for the powder or microspheres. Alternatively the powder ormicrospheres may be filled into a capsule such as a gelatin capsule, orother single dose device adapted for nasal administration. Theinsufflator can have means such as a needle to break open the capsule orother device to provide holes through which jets of the powderycomposition can be delivered to the nasal cavity.

Nasal delivery devices can be constructed or modified to dispense anoxytocin peptide or a pharmaceutical composition comprising an oxytocinpeptide wherein the oxytocin peptide or the composition is deliveredpredominantly to the inferior two-thirds of the nasal cavity. Forexample, the angle of dispersion from a delivery device such as anebulizer or an insufflator can be set so that the pharmaceuticalcomposition is mechanically directed to the inferior two-thirds of thenasal cavity, and preferably away from the superior region of the nasalcavity. Alternatively, an oxytocin peptide or a pharmaceuticalcomposition comprising an oxytocin peptide can be delivered to theinferior two-thirds of the nasal cavity by direct placement of thecomposition in the nasal cavity, for example, with a gel, an ointment, anasal tampon, a dropper, or a bioadhesive strip.

Thus in some embodiments of the disclosure, the methods compriseadministering to an individual an oxytocin peptide or pharmaceuticalcomposition comprising an oxytocin peptide wherein administration to thenasal cavity is by a nasal delivery device. The nasal delivery devicecan include, but is not limited to, unit dose containers, pump sprays,droppers, squeeze bottles, airless and preservative-free sprays,nebulizers, dose inhalers, pressurized dose inhalers, insufflators, andbi-directional devices. The nasal delivery device can be metered toadminister an accurate effective dosage amount to the nasal cavity. Thenasal delivery device can be for single unit delivery or multiple unitdelivery. In some embodiments of the disclosure, the nasal deliverydevice can be constructed whereby the angle of dispersion of apharmaceutical composition is mechanically directed towards the inferiortwo-thirds of the nasal cavity thereby minimizing delivery to theolfactory region. In some embodiments of the disclosure, the nasaldelivery device may be activated only on exhalation, thus limiting theinhalation induced and potentially undesirable distribution of thepharmaceutical composition. In some embodiments of the disclosure, thepharmaceutical composition is a gel, film, cream, ointment, impregnatedin a nasal tampon or bioadhesive strip whereby the composition is placedin the inferior two-thirds of the nasal cavity. In some embodiments ofthe disclosure, the methods include intranasal administration of anoxytocin peptide or a pharmaceutical composition comprising an oxytocinpeptide wherein the administration uses a nasal delivery device with anangle of dispersion that mechanically directs the agent to the inferiortwo-thirds of the nasal cavity wherein the oxytocin peptide isadministered after a vasoconstrictor. In some embodiments of thedisclosure, the methods include intranasal administration of an oxytocinpeptide or pharmaceutical composition comprising an oxytocin peptidewherein the administration uses a nasal delivery device with an angle ofdispersion that mechanically directs the agent to the inferiortwo-thirds of the nasal cavity wherein the oxytocin peptide isco-administered with a vasoconstrictor.

As used herein, “mucosal administration” or “administeredtransmucosally” refers to delivery to the mucosal surfaces of the nose,nasal passageways, nasal cavity; the mucosal surfaces of the oral cavityincluding the gingiva (gums), the floor of the oral cavity, the cheeks,the lips, the tongue, the teeth; and the mucosal surfaces of or aroundthe eye including the conjunctiva, the lacrimal gland, the nasolacrimalducts, the mucosa of the upper or lower eyelid and the eye.

Intranasal drug delivery has been a topic of research and developmentfor many years, although it has been only within the past decade thatcarrier systems have been devised which make delivery of substanceseffective. (Sayani and Chien (1996) Critical Reviews in Therapeutic DrugCarrier Systems, 13:85-184). Intranasal delivery has a number ofadvantageous features including comparatively high bioavailability,rapid kinetics of absorption and avoidance of a first-pass effect in theliver. In regard to patient compliance and ease of use, intranasaladministration provides a simple, rapid and non-invasive mode ofapplication. In some embodiments, intranasal administration can allowfor delivery of an oxytocin peptide to the nasal cavity and in otherembodiments, intranasal administration can allow for targeted deliveryto the trigeminal nerve. Targeted delivery to the trigeminal nerve andpreferably not the olfactory region can reduce the amount of drugentering the CNS or systemic circulation thereby reducing or eliminatingpotential undesirable CNS effects or systemic side effects. Targeteddelivery to the trigeminal nerve can also reduce the effective dosagenecessary to achieve analgesia in the facial or head regions whereinlower effective dosages will further reduce any potential CNS orsystemic side effects.

As used herein, “intranasal administration” or “administeredintranasally” refers to delivery to the nose, nasal passageways or nasalcavity by spray, drops, powder, gel, film, inhalant or other means.

The nasal cavity contains turbinate bones which protrude into the nasalcavity and generally separate it into three regions. As used herein, the“inferior region of the nasal cavity” refers to the portion of the nasalcavity where the middle and inferior turbinate bones protrude and is aregion of the nasal cavity that is innervated by the trigeminal nervesystem. The superior area of the nasal cavity is defined by the superiorturbinate bone wherein the olfactory region is located.

An oxytocin peptide is administered in a dose sufficient to provide atherapeutically effective amount to an individual to promote memoryrecall, to reduce blood glucose levels or to treat schizophrenia. Atherapeutically effective dose of an oxytocin peptide can be determinedempirically and depends on the type of treatment (e.g., reducing bloodglucose), the route of administration, and the size, weight, age andoverall health of the patient, as is within the skill of one in the artsuch as a medical practitioner.

The amount of an oxytocin peptide administered as a unit dose willdepend upon the type of pharmaceutical composition being administered,for example, a solution, a suspension, a gel, a film, an emulsion, apowder, or a sustained-release formulation. In some examples, theeffective dosage will be lower than dose amounts needed for oral,intravenous, intramuscular or subcutaneous administration, sincetransmucosal or transdermal delivery may allow for a more concentratedlevel of the oxytocin peptide within the facial and head region. Thequantity of formulation needed to deliver the desired dose will alsodepend on the concentration of the oxytocin peptide in the composition.Such determinations are within the skill of one in the art.

The therapeutic dosage of an oxytocin peptide in the pharmaceuticalcompositions used in the methods of the disclosure will depend on anumber of factors such as the chemical composition and/or modificationof the oxytocin peptide, its bioavailability by the chosen route ofadministration, its efficacy, the desired frequency of administrationcombined with the desired single dosage of the formulation and whetherthe oxytocin peptide is administered in combination with other activeagent(s). Particularly, the dosage of an oxytocin peptide will be chosento maximize memory recall or a desired blood glucose level.Pharmacological data can be obtained from animal models and clinicaltrials with normal human volunteers or patients by one with skill in theart.

As stated above, an effective amount of an oxytocin peptide will dependon the form and composition being used in the method. For example,dosages used for administration of an oxytocin peptide can include, butare not limited to, an effective amount within the dosage range of about0.1 IU to about 150 IU, or within 1 IU to about 100 IU, or within 10 IUto about 100 IU, or within about 25 IU to about 50 IU, or within about 1IU to about 40 IU, or within about 1 IU to about 30 IU, or within about4 IU to about 16 IU, or within about 4 IU to about 24 IU.

Dosages can be administered in a single dose or in multiple doses, forexample, dosages can be administered two, three, four, up to ten timesdaily depending on the type of treatment as well as on individualsusceptibility. Dosages can be administered in a sustained releaseformulation which may allow for an oxytocin peptide to be administeredless frequently such as six times a week, five times a week, four timesa week, three times a week, twice a week, or once a week, once a month,once every two months, three months, four months, five months or sixmonths or more. Infrequent administration can be accomplished bysustained release formulations.

In some embodiments of the disclosure, a composition comprising anoxytocin peptide may further comprise an additional active agent,wherein the oxytocin peptide and the additional active agent(s) areadministered as a mixture, separately and simultaneously, or separatelyin any order. In some examples the composition comprising an oxytocinpeptide is administered in combination with at least one additionalactive agent. In other examples, the composition comprising an oxytocinpeptide is administered in combination with at least two additionalactive agents.

In one embodiment, the disclosure allows for the treatment of patientswith a memory disease or disorder (e.g., Alzheimer's Disease). Inanother embodiment, the disclosure allows for the treatment of patientswith a high glucose levels.

As used herein, the term “memory improving amount”, or “glucose loweringamount,” or “effective amount” means the amount of a compositioncomprising an oxytocin peptide or analogue useful for causing animprovement in memory recall compared to memory recall prior toreceiving an effective amount of oxytocin or analogue thereof or adiminution in blood glucose levels.

The following example is provided in illustration of the disclosure andshould not be construed in any way as constituting a limitation thereof.

EXAMPLES Example 1

Participants. Subjects with a DSM-IV diagnosis of schizophrenia,confirmed by SCID interview, were enrolled in this double-blind,placebo-controlled, crossover study. Other main inclusion criteria were:minimum 18 years of age, treatment with 1 or 2 approved antipsychoticmedication with no dose changes in the previous four weeks, Positive andNegative Syndrome Scale (PANSS) score of at least 55 and a ClinicalGlobal Impressions-Severity (CGI-S) scale score of at least 4(moderately ill) at randomization. Because it was hypothesized thatoxytocin may improve paranoia due to its pro-trust effect, subjects werealso required to have a score of at least 4 (moderate) on item 6(suspiciousness/persecution) of the PANSS. This study was approved bythe UCSD IRB and written informed consent was obtained from allsubjects.

Study Drugs. Subjects were maintained on their pre-study antipsychoticmedication regimen and doses were not changed during the study. Subjectsreceived 3-weeks of daily intranasal oxytocin (Syntocinin, Novartis,Basel Switzerland) and 3-weeks of daily intranasal placebo. Oxytocin wasdosed at 20 IU (5 sprays) twice a day for the first week and 40 IU (10sprays) twice a day thereafter. Order of treatment (placebo-oxytocin oroxytocin-placebo) was randomly assigned using a computer generatedrandom sequence.

Efficacy and Safety assessments. Subjects, raters and study staffenrolling patients were blinded to treatment condition. The total studyduration for each individual subject was 7 weeks. Subjects wereevaluated 7 times. Visit 1 and 5 were baseline assessments and visits 2,3, 4 and 6, 7, 8 were the weekly visits for the two treatment periods,respectively. Washout occurred in the week between visit 4 and 5. Ateach visit raters assessed subjects utilizing the PANSS, CGI-S andCGI-Improvement (CGI-I). PANSS-total at the final visit of eachtreatment period was chosen a′ priori as the primary efficacy endpoint.

Safety was assessed at each visit by a medical exam, and assessments ofreported adverse events. In addition, urine was collected for osmolalitytesting and blood was drawn at each visit and analyzed at UCSDlaboratories for basic chemistry analysis. Statistical Methods. Datafrom all subjects who received at least one dose and one assessment inboth treatment periods (intent-to-treat population) were subjected toanalysisusing SPSS version 11.0. Baseline scores for both drugtreatments were compared for similarity using paired t-test. The changein baseline scores from period 1 to period 2 was compared among theplacebo-oxytocin versus oxytocin-placebo group using a two-sample t-testto assess for period carry-over effects. PANNS and CGI data weresubjected to repeated measures ANOVA with drug and treatment-week asrepeated measures factors. Treatment sequence was included as abetween-subjects factor to evaluate possible period and carry-overeffects. Paired t-tests, corrected for multiple-comparisons usingBonferoni method were used to compare placebo and oxytocin scores atendpoint and each the other assessment visits. Cohen's d statistic wascalculated for each measure at endpoint.

Fifteen of 19 randomized subjects completed all study visits. Fourrandomized subjects were discontinued before completing the study, onedue to nasal discomfort from the intranasal sprays, 3 due toinsufficient compliance. None of the discontinued subjects reached thesecond treatment period, therefore, intent-to-treat population wasidentical to completer population. Eighty percent of completers weremale, 53% black, 27% white, average age was and duration of disease was48 (8.9) and 26 (14.6), respectively (Table 1). They were on a widerange of antipsychotics entering the study (table 2).

TABLE 1 Demographics of completers N % Gender Male 12 80% Female 3 20%Race Caucasian 4 26.70% Black 8 53.30% Other 3 20% Initial TreatmentOxytocin 6 40% Placebo 9 60% Age (STD) 48 (8.9) Average years ill 25.8(14.5) (STD)

TABLE 2 Antipsychotics Subjects Received Prior to and During Study Drugtype N Quetiapine 6 Aripiprazole 4 Risperidone 4 Olanzapine 2Ziprasidone 1 Chlorpromazine 1

Table 3 lists efficacy results. Baseline scores at the start of eachplacebo and oxytocin treatment arm were highly similar for PANSS-total(82.1±11.06 and 81.4±12.43) and CGI-S (4.60±0.74 and 4.67±0.74). Changein scores between first period and second period baselines did notsignificantly differ between groups, suggesting there wasn't significantcarry-over.

TABLE 3 Efficacy Scores (±Standard Deviation) Week 3 Baseline Week 1Week 2 [Cohen's d]^(#) PANSS Total Placebo 82.1 (11.1) 76.9 (10.6) 75.7(12.7) 79.1 (12.9) Oxytocin 81.5 (12.4) 76.3 (11.3) 76.9 (13.2) 73.6(13.6)** [0.43] PANSS Positive Placebo 22.8 (5.2) 21.2 (5.0) 20.0 (4.6)21.9 (4.8) Oxytocin 21.7 (4.1) 20.6 (4.5) 20.5 (4.6) 19.9 (5.2)** [0.40]PANSS Negative Placebo 21.8 (4.7) 20.5 (4.4) 20.2 (4.6) 20.7 (4.3)Oxytocin 20.2 (4.7) 20.1 (4.8) 19.7 (4.3) 18.5 (4.5)* [0.50] PANSS Gen.Psy. Placebo 37.5 (6.6) 35.2 (6.2) 35.4 (7.3) 36.4 (7.3) Oxytocin 38.8(7.6) 36.4 (8.1) 36.3 (8.2) 34.8 (6.9) [0.24] PANSS CGI-I Placebo 4.60(0.74){circumflex over ( )} 3.53 (0.92) 3.53 (0.92) 3.73 (1.03) Oxytocin4.67 (0.61){circumflex over ( )} 3.33 (0.62) 3.33 (1.20) 3.07 (0.70)**[0.74] Significantly different Vs. Placebo *P < 0.05, **P < 0.01 ^(#)0.2small, 0.5 medium, 0.8 large; {circumflex over ( )}CGI-Severity and notCGI-Improvement noted for baseline

There was a significantly greater improvement in PANSS-total scoresacross visits with oxytocin compared to placebo as revealed by asignificant Drug-by-Treatment Week interaction (F(2,26)=6.493, p=0.005).None of the other main or interaction effects were significant. Scoreswere significantly lower with oxytocin versus placebo at the endpoint(week 3) visit (diff.=5.46, p<0.001, Cohen d effect size=0.43) whereasthere was no significant difference at baseline (difference=0.60), week1 (difference=0.60) or week 2 (difference=1.20).

CGI-I scores revealed a Drug-by-Treatment Week interaction thatapproached significance (P=0.065, Cohen's d=0.74). No other main orinteraction effect was significant. CGI-I was significantly lower foroxytocin versus placebo at week 3 (P<0.001) but not at baseline, week 1,or week 2.

Analysis of PANSS negative subscale scores did not reveal anysignificant main factor or interaction effects but were significantlylower for oxytocin versus placebo at week 3 only (p=0.023, Cohen'sd=0.50).

A greater decrease in PANSS positive subscale scores under oxytocin wasreflected in a non-significant trend toward a Drug-by-Treatment Weekinteraction (p=0.089). There was also a significant Drug by TreatmentSequence effect (F(1.13)=11.57, p=0.005) reflected in the fact thatPANSS positive scores were significantly lower with oxytocin treatment(P=0.01) when it was the first treatment but not when it was the secondtreatment. There were no other significant main or interaction effects.Oxytocin scores were significantly lower than placebo scores at week 3(P=0.006, Cohen's d=0.4) but not at baseline, week 1 or week 2.

A greater decrease in PANSS general psychopathology subscale underoxytocin was reflected in a non-significant trend toward a Drug-by-Visitinteraction (p=0.069, Cohen's d=0.24). Oxytocin scores were notsignificantly different than placebo at any time point.

Overall, differences in the first period by itself (between subjectsanalysis) did not reach statistical significance.

There were no serious adverse events reported during the study and nosignificant differences in rates of reported adverse effects withoxytocin compared to placebo (Table 4), There was no significantdifferences between oxytocin and placebo in any of the measured bloodchemistry or urine osmolality (Table 5).

TABLE 4 Reported Adverse Events Oxytocin (n) Placebo (n) Headache 26.7%(4) 20.0% (3) Dyspepsia or nausea 26.7% (4) 40.0% (6) Sleep impairment33.3% (5) 26.7% (4) Dizzy or lightheaded 26.7% (4) 20.0% (3) NasalIrritation 26.7% (4) 13.3% (2) Lethargy  0.0% (0) 13.3% (2)

TABLE 5 Chemistry analysis at endpoint Urine Glucose BUN CreatinineBicarb. Chloride Sodium Potassium Calcium Osmolality (mg/dL) (mg/dL)(mg/dL) (mmol/L) (mmol/L) (mmol/L) (mmol/L) (mg/dL) (mOsm/kg) Oxytocin112.5 10.8 0.9 27.9 101.1 138.5 4.4 9.3 607.9 Placebo 128.9 10   0.927.4 100.8 138.5 4.2 9.3 557.6

The disclosure demonstrates that 3 weeks of intranasal oxytocin, givenadjunctive to standard antipsychotic medications, caused significantlygreater reductions in schizophrenia symptoms at the study endpointcompared to placebo. This result supports the hypothesis that oxytocinexhibits antipsychotic properties.

Oxytocin's effect appeared to manifest broadly across symptom clustersincluding positive and negative symptoms, although the improvement inpositive symptoms appeared statistically more robust. Though thenumerical effect of oxytocin is modest (7.9 point reduction on thePANSS-total compared to 3.0 points for placebo), three points regardingits observed magnitude of benefit warrant consideration. First, subjectswere already on stable therapeutic doses of at least one antipsychoticand oxytocin represented adjunctive treatment. Compared to amedication-free cohort, improvements in this already-treated cohort aregenerally harder to come by. Notwithstanding this fact, the effect sizeof the improvements observed were medium ((0.43) PANSS scores) to large((0.74) CGI-I). Second, this study had dosing and duration limitationsthat may have prevented the optimal magnitude of benefit from beingobserved: only one oxytocin dose was studied, and subjects were treatedfor only 3 weeks. Either higher doses or longer treatment duration mayhave yielded greater symptom improvements. Supporting this latter point,oxytocin's benefits emerged only at the week 3 assessment, a findingthat suggests a delayed onset of action which may have grown with longertreatment duration. Finally, while it is possible that a certainsubpopulation of schizophrenia patients is particularly responsive tothe benefits of oxytocin (based on receptor variations or diagnosticsubtype), the sample size was too small for a subanalysis along theselines. To fully characterize oxytocin's antipsychotic potential, futurestudies are warranted with larger sample sizes, different doses, longertreatment durations, as well as pharmacogenetic and behavioralinvestigations.

Despite its therapeutic potential, there have been very few trials ofoxytocin for psychiatric conditions. As such, the finding thatoxytocin—given twice daily for three weeks—was well-tolerated and didnot appear to produce any subjective or objective adverse events isnoteworthy.

The disclosure demonstrates that intranasal oxytocin, given adjunctiveto standard antipsychotic medications, caused significantly greaterreductions in schizophrenia symptoms at the study end point comparedwith placebo. In addition, the intranasal administration demonstrated animprovement in memory and a reduction in blood glucose levels comparedto placebo.

Example 2

Participants. Subjects with a DSM-IV diagnosis of schizophrenia,confirmed by SCID interview, were enrolled in a double-blind,placebo-controlled, crossover study. Other main inclusion criteria were:minimum 18 years of age, treatment with 1 or 2 approved antipsychoticmedication with no dose changes in the previous four weeks, a minimumPositive and Negative Syndrome Scale (PANSS) score of at least 55 with aminimum score of 4 and a Clinical Global Impressions-Severity (CGI-S)scale score of at least 4 (moderately ill) at randomization.

Study Drugs. Subjects were maintained on a stable dose of theirpre-study antipsychotic medication regimen. In addition to theirantipsychotics, subjects received 3-weeks of daily intranasal oxytocin(Syntocinin, Novartis, Basel Switzerland) dosed 20 IU twice a day forthe first week and then 40 IU twice a day for weeks two and three, and3-weeks of daily intranasal placebo. Order of treatment(placebo-oxytocin or oxytocin-placebo) was randomly assigned using acomputer generated random sequence.

Safety and efficacy assessments. Subjects, raters, and study staffenrolling patients were blinded to treatment condition. The total studyduration for each individual subject was 7 weeks, which included abaseline visit (V1), a three-week initial treatment period, a 1 weekwashout (between V4 and V5), a repeat baseline visit, and a three-weekcrossover treatment period. Subjects received the cognitive tests atotal of 3 times: at baseline visits (V1, and after 3 weeks of treatmentwith placebo and oxytocin (V4, V7).

The CVLT-II is a commonly-used test of new learning and declarativeverbal memory, which closely resembles the HVLT (Hopkins Verbal LearningTest), a part of the standardized MATRICS cognitive battery, now used tomeasure cognitive changes in Schizophrenia clinical trials. In theCVLT-II, lists of 16 words (List A) from various semantic categories areread over 5 trials. After each trial, subject is asked to recall as manywords as they can (free recall). An interference list (List B) is thenpresented. Free and cued recall of List A is measured immediatelyfollowing (short delay) and 20 minutes (long delay). At the end of thetest, a 44 word recognition task is performed with subjects identifyingtarget words among 28 distractors. The CVLT's format allows assessmentof multiple aspects of cognition: overall recall ability, rate oflearning over sequential trials, ability to retain learned material, anditem recognition.

The Letter Number Sequencing subtest (LNS) is a component of the largerWAIS-III and is used to measure attention and working memory. It issimilar to the Letter number span test, which is also part of theMATRICS. In each trial of the LNS, a list of letters and numbers areread in a mixed order to participants; participants are asked to recallfirst the digits and then the letters in the order they were presented.Length of the list ranges from 2 to 8; each length is tested threetimes. Scores are calculated by totaling the correct responses for eachlength.

For each subtest a two-factor repeated ANOVA with drug treatment(placebo/oxytocin) and order of treatment as factors was conducted.

Of the 20 subjects enrolled, 15 (12 males, 3 females) completed bothtreatment arms and thus provided CVLT and LNS data on both placebo andoxytocin. Average age was 48.0+/−8.9 years and had been ill an averageof 25.8+/−14.5 years.

Subjects scored higher on all 9 subscales of the CVLT after 3 weeks ofoxytocin than after placebo (Table 6). Oxytocin outperformed placeboregardless of the order oxytocin was given, obviating the possibility ofa ‘practice’ effect.

TABLE 6 CLVT Data Baseline Placebo Oxytocin Mean Mean Mean Test (S.E.M.)(S.E.M) (S.E.M.) Trials 1-5 Raw 45.0 (3.00) 51.33 (4.51) 55.67 (3.65)Short Delay Free 8.27 (0.88)  9.47 (1.22) 10.60 (1.03) Recall Raw ShortDelay Cued 9.87 (0.87) 10.87 (1.07) 12.00 (0.79) Recall Raw Long DelayFree 9.07 (0.96) 10.73 (0.85) 10.87 (0.87) Recall Raw Long Delay Cued10.0 (0.87) 11.33 (1.04) 11.87 (0.97) Recall Raw Total Repetitions 8.73(2.74) 10.07 (4.71) 13.67 (5.44) Raw Total Intrusions 6.53 (2.08)  6.00(1.94)  4.93 (1.90) Raw Total Recall 1.77 (0.17)  2.05 (0.21)  2.22(0.19) Discriminability Raw Total Recognition 2.79 (0.21)  2.63 (0.36) 2.97 (0.28) Discriminability Raw

In contrast to the CVLT findings, there was no significant difference onany of the scores on the LNS between oxytocin and placebo.

The disclosure provides evidence of beneficial effects of oxytocin onnon-social cognition. Contrary to the original concern that oxytocinmight have a negative effect on memory and learning, no deleteriouseffect of 3-weeks of daily oxytocin on either of the cognitive tests wasobserved. Surprisingly, oxytocin enhanced performance on all CVLT with astronger enhancement of short-delay memory over long-delay memory.

It is noteworthy that the well-known “practice effect” phenomenonwhereby repeat administration of cognitive tests produces enhancedperformance was masked in this study by drug treatment effects. That is,the expected improvement in performance in the last test session (end ofsecond treatment period) compared to the previous test administration(end of first treatment period) was only seen among subjects whoreceived oxytocin when the last test was administered. Subjects whoreceived placebo when the last test was administered exhibited adecrement in scores compared to their previous testing while receivingoxytocin.

In contrast to CVLT results, oxytocin did not have an effect on the LNS.Though this negative finding is important in that it addresses concernsthat that repeated daily oxytocin administration may have a negativeeffect on working memory in patients with schizophrenia, it contraststhe positive effects of oxytocin seen in the CVLT. One possibleexplanation for these discrepant effects involves the different aspectsof cognition assayed by the different tests. The CVLT is a test ofverbal learning that uses whole words and therefore probes deeperlexical content, whereas the LNS is a less meaning-based measure ofworking memory using letters and digits, and invoking little semanticprocessing.

In studies using socially salient stimuli such as words relevant torelationship, reproduction or sex or photos of faces, the results havegenerally found a mnemonic effect in normal subjects after a single doseof oxytocin (24 IU) but at least one study found the opposite effectwith socially-relevant words. In contrast previous studies have shownthat when measuring memory for non-socially relevant stimuli, oxytocinhas no effect or even worsens performance.

Because the CVLT uses neutral words (furniture”, “vegetables”, “ways oftraveling”, and “animal”) but not social, sexual, orreproduction-related words the mnemonic effect of oxytocin in this studystands in contrast to previous findings, which found no effect or aworsening of memory for non-socially relevant stimuli.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the methods, treatments and compositions ofthe disclosure, and are not intended to limit the scope of what theinventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims.

The invention claimed is:
 1. A method for reducing blood glucose levelsof a subject, the method comprising intranasally administering to thesubject a formulation comprising an oxytocin peptide or analogue thereofat least once per day at a dose of about 20-100 IU, wherein the dose ofoxytocin peptide or analogue thereof reduces a subject's blood glucosecompared to the subject's blood glucose before administration of theoxytocin peptide or analogue thereof.
 2. The method of claim 1, whereinthe total daily dose is about 100 IU per day.
 3. The method of claim 1,wherein the dose is at least two times per day.
 4. The method of claim1, wherein the dose is about 40-80 IU per day.